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Abstract:

Disclosed are devices for holding samples, particularly liquid samples,
during high-pressure treatment. The devices enable a variety of
functions, such as high-throughput screening of samples in
multi-compartment device embodiments, and adjustment of solution
conditions during high-pressure treatment. The devices are designed to
maintain integrity during the high-pressure conditions, and are
optionally substantially impermeable to oxygen.

Claims:

1. A container for pressure treatment of a liquid sample, comprising at
least one compartment for holding the liquid sample, wherein said
container is fabricated from a flexible material, where the material can
withstand up to 10 kbar of multi-dimensional pressure without breakage or
rupture and is optionally substantially impermeable to oxygen at high
pressure.

2. The container of claim 1, wherein the container has a variable loading
volume at standard pressure.

3. The container of claim 2, wherein the container comprises a cylinder,
said cylinder having a first end and a second end; a moveable plug
inserted into the first end of the cylinder; and a removable portion
affixed to the second end of the cylinder.

4. The container of claim 3, wherein the removable portion is a threaded
screw-cap.

5. The container of claim 3, wherein the removable portion is a tip that
can be cut off of the container or broken off of the container.

7. A method of subjecting a sample to high pressure, comprising
introducing a sample into the container of claim 1, subjecting the
container to high pressure, and reducing the pressure to atmospheric
pressure.

8. The method of claim 7, wherein the sample is a solution of an
aggregated protein and/or a denatured protein.

9. The container of claim 1, wherein the container is formed from a
material selected from the group consisting of polyethyleneterephthalate,
high-density polyethylene, polystyrene, and polystyrene-butadiene block
copolymers.

10. The container of claim 1, wherein the container has a constant loading
volume at standard pressure.

11. A device for solution exchange at high pressure, comprising:at least
one first container holding a first liquid sample;one or more additional
containers holding an additional liquid sample or samples, where the
first liquid sample and additional liquid sample or samples can be the
same or different,where the containers are fabricated from materials that
can withstand up to 5 kbar of pressure without breakage or rupture and
optionally are substantially impermeable to oxygen at high pressure,and
where the liquid sample of the one or more additional containers can be
mixed or contacted with the liquid sample of the first container, while
both first and additional containers and their respective liquid samples
can be maintained at high pressure before, during, and after mixing or
contacting.

12. The device of claim 11, wherein when the one or more additional
containers comprise two or more additional containers, the contents of
the two or more additional containers can be mixed with the contents of
the first container either independently of the other two or more
additional containers, or in conjunction with the other two or more
additional containers.

13. The device of claim 11, wherein:the at least one first container is a
pre-mix container holding a liquid sample (where the liquid samples can
be the same or different);the one or more additional containers holding
an additional liquid sample or samples is/are another pre-mix container,
where the first liquid sample and additional liquid sample or samples can
be the same or different,and the device further comprises at least one
additional receiving container, where the receiving container can be
empty prior to transfer or can contain a liquid or solid composition
prior to transfer;where all the containers are fabricated from materials
that can withstand up to 5 kbar of pressure without breakage or rupture
and optionally are substantially impermeable to oxygen at high
pressure;where the liquid samples in the pre-mix containers holding
liquid samples can be transferred into the at least one receiving
container whereby the liquid samples can contact and/or mix with each
other;and where the pre-mix containers holding liquid samples, the at
least one receiving container, and the liquid samples themselves can be
maintained at high pressure before, during, and after contacting and/or
mixing.

14. The device of claim 13, further comprising a mixing device interposed
in the fluid path between the pre-mix containers holding liquid samples
and the at least one receiving container.

15. The device of claim 14, wherein the mixing device is a static mixer.

16. The device of claim 11, where the first container comprises a
compartment for holding a first liquid sample, the first container is
fabricated from a flexible material that can withstand up to 5 kbar of
pressure without breakage or rupture and optionally is substantially
impermeable to oxygen at high pressure;and one or more additional
containers, where the one or more additional containers are fabricated
from a flexible material that can withstand up to 5 kbar of pressure
without breakage or rupture and optionally is substantially impermeable
to oxygen at high pressure;where the one or more additional containers
are completely enclosed by the first container, and where the one or more
additional containers contains additional liquid samples, which can be
the same or different from each other and from the first liquid
sample;where the one or more additional containers can be opened while
within the first container, whereby the first liquid sample and
additional liquid sample(s) can contact and/or mix.

17. The device of claim 16, wherein:the one or more additional containers
comprise a cap(s) which can be maintained in a closed position, where the
cap can be opened without opening the first container;and while the first
container, one or more additional containers, and all liquid samples can
be maintained at high pressure before, during, and after opening the
cap(s) of the one or more additional containers.

18. The device of claim 17, wherein the cap is also capable of mixing the
liquid sample contained in the first container with the liquid samples of
the one or more additional containers.

19. The device of claim 17, wherein the cap(s) comprises a magnetized
portion, such as a magnetic disk.

20. The device of claim 11, wherein the at least one first container and
the one or more additional containers are connected in a flow loop.

21. The device of claim 20, further comprising a check valve whereby fluid
can only flow in one direction in the loop.

22. The device of claim 11, wherein one or more solution conditions of the
at least one first liquid sample in the at least one first container are
changed when the liquid of the at least one first container is mixed
and/or contacted with the liquid in the at least one additional
container.

24. A method of altering solution conditions while under high pressure,
comprising:providing at least one first container holding a first liquid
sample;providing one or more additional containers holding an additional
liquid sample or samples, where at least one of the at least one first
liquid sample and additional liquid sample or samples is different from
the remaining samples;where the containers are fabricated from materials
that can withstand up to 5 kbar of pressure without breakage or rupture
and optionally are substantially impermeable to oxygen at high
pressure,and mixing or contacting the liquid sample of the one or more
additional containers with the liquid sample of the first container,
thereby altering the solution conditions of the at least one first liquid
sample;while maintaining high pressure before, during, and after mixing
or contacting.

25. The method of claim 24, wherein the one or more solution conditions of
the at least one first liquid sample are selected from: pH, salt
concentration, reducing agent concentration, oxidizing agent
concentration, both reducing agent concentration and oxidizing agent
concentration, chaotrope concentration, arginine concentration,
surfactant concentration, preferentially excluding compound
concentration, ligand concentration, concentration of any compounds
originally present in the solution, or addition of another reactant or
reagent.

26. The method of claim 25, wherein the one or more solution conditions of
the at least one first liquid sample is pH.

27. The method of claim 26, wherein the pH of the at least one first
liquid sample is about pH 9 to about pH 11 before solution exchange, and
the pH of the at least one first liquid sample is about pH 7 to about pH
8.9 after solution exchange is complete.

28. The method of claim 27, wherein the pH of the at least one first
liquid sample is changed in a step-wise fashion.

29. A multi-sample holding device comprising at least two compartments for
receiving liquid samples, wherein said device maintains the compartments
as substantially closed systems when subjected to high pressure.

30. A multi-sample holding device comprising:a) a body made from a
material that maintains integrity under high pressure; andb) a plurality
of sample compartments in the body, adapted for receiving liquid
samples;wherein the device does not permit significant transfer of liquid
sample either between the plurality of sample compartments or between any
sample compartment and the surroundings.

32. The device of claim 30, wherein the body is formed from a material
selected from the group consisting of polyethyleneterephthalate,
high-density polyethylene, polystyrene, and polystyrene-butadiene block
copolymers.

[0002]This invention pertains to devices, such as containers, multiwell
plates, and systems for pumping fluids to containers and multiwell
plates, designed for operation at high hydrostatic pressure. The
invention also pertains to methods of using the devices for refolding of
proteins under high pressure.

BACKGROUND

[0003]Many proteins are valuable as therapeutic agents. Such proteins
include human growth hormone, which is used to treat abnormal height when
insufficient growth hormone is produced in the body, and
interferon-gamma, which is used to treat neoplastic and viral diseases.
Protein pharmaceuticals are often produced using recombinant DNA
technology, which can enable production of higher amounts of protein than
can be isolated from naturally-occurring sources, and which avoids
contamination that often occurs with proteins isolated from
naturally-occurring sources.

[0004]Proper folding of a protein is essential to the normal functioning
of the protein. Improperly folded proteins are believed to contribute to
the pathology of several diseases, including Alzheimer's disease, bovine
spongiform encephalopathy (BSE, or "mad cow" disease) and human
Creutzfeldt-Jakob disease (CJD), and Parkinson's disease; these diseases
serve to illustrate the importance of proper protein folding.

[0005]Several proteins of therapeutic value in humans, such as recombinant
human growth hormone and recombinant human interferon gamma, can be
expressed in bacteria, yeast, and other microorganisms. While large
amounts of proteins can be produced in such systems, the proteins are
often misfolded, and often aggregate together in large clumps called
inclusion bodies. The proteins cannot be used in the misfolded,
aggregated state. Accordingly, methods of disaggregating and properly
refolding such proteins have been the subject of much investigation.

[0006]One method of refolding proteins uses high pressure on solutions of
proteins in order to disaggregate, unfold, and properly refold proteins.
Such methods are described in U.S. Pat. No. 6,489,450, U.S. Patent
Application Publication No. 2004/0038333, and International Patent
Application WO 02/062827. Those disclosures indicated that certain
high-pressure treatments of aggregated proteins or misfolded proteins
resulting in recovery of disaggregated protein retaining biological
activity (i.e., the protein was properly folded, as is required for
biological activity) in good yields. U.S. Pat. No. 6,489,450, U.S.
2004/0038333, and WO 02/062827 are incorporated by reference herein in
their entireties.

[0007]As indicated in U.S. 2004/0038333, empirical screening procedures
are sometimes required to determine the optimal refolding conditions for
a protein. Thus there is a need for suitable equipment which can be used
in methods to rapidly determine the optimal conditions, such as multiwell
plates, disposable single-sample containers, and devices for mixing
solutions under high pressure in order to change solution conditions
under high pressure.

[0008]96-well plates (typically with an 8×12 arrangement of wells)
are commonly used in high-throughput screening in biology and
biochemistry. However, current commercially available plates are not
suitable for high-pressure applications (e.g., 250 bar and higher). The
present invention provides such equipment which is suitable for use under
high pressure.

[0009]Single-sample containers currently used for high-pressure studies
also suffer from drawbacks. Containers made from materials such as
low-density polyethylene and polypropylene allow significant mass
transfer of oxygen under high pressures. For reactions which are
sensitive to the redox environment of the solution, such oxygen transfer
is highly undesirable. The present invention also provides equipment
which reduces or eliminates oxygen mass transfer through the walls of the
container when desired.

[0010]Yet another drawback of currently used equipment is that solution
conditions cannot be adjusted during the high pressure treatment. The
present invention provides equipment which allows changes in solution
conditions during high pressure treatment, by enabling manipulation of
various containers and solutions while the containers and solutions are
inside the high pressure apparatus.

DISCLOSURE OF THE INVENTION

[0011]The invention embraces single-sample holding devices, multi-sample
holding devices, and solution exchange devices suitable for use at high
pressure. In certain embodiments, the devices are fabricated from
polymers, which allows relatively low cost fabrication of the devices.
This also allows for injection molding of the devices for convenient
fabrication. In certain embodiments, the devices can be disposable for
ease of use. The solution exchange devices permit changing of solution
conditions of the sample while the sample is maintained under high
pressure. In optional embodiments, the devices can be made from materials
which are substantially oxygen-impermeable.

[0012]In one embodiment, the invention embraces a multi-sample holding
device comprising at least two compartments for receiving liquid samples,
wherein the device maintains the compartments as substantially closed
systems when subjected to high pressure.

[0013]In another embodiment, the invention embraces a multi-sample holding
device comprising: a) a body made from a material that maintains
integrity under high pressure; and b) a plurality of sample compartments
in the body, adapted for receiving liquid samples; wherein the device
does not permit significant transfer of liquid sample either between the
plurality of sample compartments or between any sample compartment and
the surroundings.

[0015]In one embodiment of the multi-sample holding devices, the sample
compartments have openings on the top side of the device, and the
openings of the sample compartments are sealed by placing a sealing mat
on top of the device so as to cover the openings of the sample
compartments. The sealing mat can be maintained in place by a
constant-tension clamp. In another embodiment of the multi-sample holding
device, the sample compartments are sealed by placing heat-sealed septums
in the openings of the compartments prior to loading the compartments
with samples. The samples can be loaded via needle injection through the
septums. An adhesive polymeric membrane can then be placed on top of the
device and septums to ensure adequate sealing.

[0016]In further embodiments of the foregoing multi-sample holding
devices, the body of the devices is formed from a material selected from
the group consisting of polyethyleneterephthalate, high-density
polyethylene, polystyrene, and polystyrene-butadiene block copolymers. In
another embodiment of the foregoing multi-sample holding devices, the
body is formed from polyethylene-terephthalate. In another embodiment of
the foregoing multi-sample holding devices, the body is formed from
polystyrene-butadiene block copolymers.

[0017]In another embodiment, the invention embraces a container for
pressure treatment of a liquid sample, where the container comprises at
least one compartment for holding the liquid sample, where the container
is fabricated from a flexible material, where the material can withstand
up to about 5 kbar, preferably up to about 10 kbar of pressure without
breakage or rupture and optionally is substantially impermeable to oxygen
at high pressure. (The pressure indicated is a multidimensional pressure
on the entire container, not a differential pressure.) In one embodiment,
the container has only one compartment for holding the liquid sample. In
one embodiment, the container has a constant loading volume at standard
pressure. In another embodiment, the container has a variable loading
volume at standard pressure.

[0018]In another embodiment, the container having a variable loading
volume comprises a cylinder having a first end and a second end. A
moveable plug is inserted into the first end of the cylinder; and a
removable portion is affixed to the second end of the cylinder which can
be detached to allow removal of the contents of the cylinder. The
removable portion can be a cap, which can be threaded and engage with
complementary threads on the second end of the cylinder, or can snap on,
or can be affixed magnetically. In another embodiment, a short narrow
protrusion extends from the second end of the cylinder, bearing threads
or other methods of engaging a cap; the cap is placed on the protrusion,
for later removal to allow removal of the contents of the cylinder. In
one embodiment, the narrow protrusion can bear Luer-Lok® fittings
(Luer-Lok® is a registered trademark of Becton, Dickinson & Co.,
Franklin Lakes, N.J. for an interlocking connection system).

[0019]In another embodiment, the container having a variable loading
volume comprises a cylinder having a first end and a second end. A
moveable plug inserted into the first end of the cylinder. A sealed tip
is attached to the second end of the cylinder which can be detached to
allow removal of the contents of the cylinder. The sealed tip can be a
short narrow protrusion from the second end of the cylinder which can be
broken off to allow removal of the contents of the cylinder. In some
embodiments, the tip can be broken off manually; in other embodiments,
the tip cannot be broken off manually and is broken off using a cutting
tool.

[0020]In another embodiment, the moveable plug for use in the variable
volume loading container has a one-way valve. The one-way valve plug
allows air and sample within the container to be bled out at standard
pressure, while preventing flow back through the valve into the container
of any air, gas, or liquid from outside the container. In one embodiment,
the one-way valve is a check valve. In another embodiment, the one-way
valve is a ball check valve. In another embodiment, the one-way valve is
a ball-and-spring check valve. In another embodiment, the one-way valve
is a flap check valve. In another embodiment, the one-way valve is a duck
bill backflow valve. In another embodiment, the one-way valve is an
umbrella valve. In another embodiment, the one-way valve is a swing-check
valve. In another embodiment, the one-way valve is a lift-check valve.

[0021]In further embodiments of the foregoing containers, the container is
formed from a material selected from the group consisting of
polyethyleneterephthalate, high-density polyethylene, polystyrene, and
polystyrene-butadiene block copolymers. In another embodiment of the
foregoing containers, the container is formed from
polyethylene-terephthalate. In another embodiment of the foregoing
containers, the container is formed from polystyrene-butadiene block
copolymers.

[0022]In another embodiment, the invention embraces a system for solution
exchange (solution mixing) under pressure, comprising a first container
holding a first liquid sample and one or more additional containers
holding an additional liquid sample or samples, where the first liquid
sample and additional liquid sample or samples can be the same or
different, where the containers are fabricated from materials that can
withstand up to about 5 kbar, preferably up to about 10 kbar of pressure
(multidimensional pressure on the system, not a differential pressure)
without breakage or rupture and optionally are substantially impermeable
to oxygen at high pressure, and where the liquid sample of the one or
more additional containers can be mixed with the liquid sample of the
first container while both first and additional containers and their
respective liquid samples can be maintained at high pressure before,
during, and after mixing. When the one or more additional containers
comprise a plurality of containers, i.e., two or more additional
containers, the contents of the two or more additional containers can be
mixed with the contents of the first container either independently of
the other two or more additional containers (i.e., at different times),
or in conjunction with the other two or more additional containers (i.e.,
simultaneously or in a pre-determined time series). The high pressure
before mixing or contacting, the high pressure during mixing or
contacting, and the high pressure after mixing or contacting can all be
the same pressure, or two can be the same pressure and one can be
different pressures, or all three pressures can be different pressures.

[0023]In another embodiment of the system for solution exchange (solution
mixing) under pressure, the system comprises at least two pre-mix
containers holding liquid samples (where the liquid samples can be the
same or different) which are designated the pre-mix containers, and at
least one additional container designated the receiving container, where
the receiving container can be empty prior to transfer or can contain a
liquid or solid composition prior to transfer, where the containers are
fabricated from materials that can withstand up to about 5 kbar,
preferably up to about 10 kbar of pressure (multidimensional pressure on
the system, not a differential pressure) without breakage or rupture and
optionally are substantially impermeable to oxygen at high pressure,
where the liquid samples in the at least two pre-mix containers holding
liquid samples can be moved into the at least one receiving container
where the liquid samples can contact each other, and the at least two
pre-mix containers holding liquid samples, the at least one receiving
container, and the liquid samples themselves can be maintained at high
pressure before, during, and after mixing. In one embodiment, a mixing
device, such as a static mixer (such as those used for HPLC solvent
mixing), can be interposed in the fluid path between the at least two
pre-mix containers holding liquid samples and the at least one receiving
container in order to facilitate mixing of the liquid samples. In other
embodiments, flow from one or more of the pre-mix containers can be
controlled independently by valves, to allow contents from certain
pre-mix containers to be drawn into the receiving container, while
preventing flow from other selected pre-mix containers; at a later
period, the valves can be set to permit the contents of the other
selected pre-mix containers to flow into the receiving container.

[0024]In another embodiment of the system for solution exchange (solution
mixing), the invention comprises a first container, where the first
container comprises a compartment for holding a first liquid sample, the
first container is fabricated from a flexible material that can withstand
up to about 5 kbar, preferably up to about 10 kbar of pressure
(multidimensional pressure on the system, not a differential pressure)
without breakage or rupture and optionally is substantially impermeable
to oxygen at high pressure; and one or more additional containers, where
the one or more additional containers are fabricated from a flexible
material that can withstand up to about 5 kbar, preferably up to about 10
kbar of pressure (multidimensional pressure on the system, not a
differential pressure) without breakage or rupture and optionally is
substantially impermeable to oxygen at high pressure, and where the one
or more additional containers are completely enclosed by the first
container, and where the one or more additional containers contains
additional liquid samples, which can be the same or different from each
other and from the first liquid sample; where the one or more additional
containers can be opened while within the first container (either
independently of the other additional containers, or in concert with the
additional containers), allowing the first liquid sample and additional
liquid samples to mix. In one embodiment, the one or more additional
containers comprise a cap which can be maintained in a closed position,
where the cap can be opened without opening the first container, and
while the first container, one or more additional containers, and all
liquid samples can be maintained at high pressure before, during, and
after mixing. In another embodiment, the cap is also capable of mixing
the liquid sample contained in the first container with the liquid
samples of the one or more additional containers. In another embodiment,
the cap comprises a magnetized portion, such as a magnetic disk.

[0025]In another embodiment of the system for solution exchange (solution
mixing), the invention embraces a container system for pressure treatment
of a liquid sample, comprising a first container which comprises a
compartment for holding the liquid sample, where the first container is
fabricated from a flexible material, where the material can withstand up
to about 5 kbar, preferably up to about 10 kbar of pressure
(multidimensional pressure on the system, not a differential pressure)
without breakage or rupture and optionally is substantially impermeable
to oxygen at high pressure; and also comprising at least one additional
container, where the at least one additional container is fabricated from
a flexible material, where the material can withstand up to about 5 kbar,
preferably up to about 10 kbar of pressure (multidimensional pressure on
the system, not a differential pressure) without breakage or rupture and
optionally is substantially impermeable to oxygen at high pressure, and
where the first container and the at least one additional container are
connected by a flow loop. The flow loop comprises a check valve which
permits flow in the flow loop in only one direction, and a pump capable
of operating when the container system is subjected to high pressure. The
pump can be controlled by a microprocessor. When the microprocessor is
included within the high pressure apparatus, it can be powered by a
battery which is also included within the high pressure apparatus, or by
power lines which are run into the high pressure apparatus.
Alternatively, the device can be controlled by drive shafts which enter
the high pressure chamber through appropriately sealed openings into the
high pressure chamber. The flow loop can bypass one or more of the one or
more additional containers via bypass shunts; valves can close the bypass
shunts and connect the one or more additional containers to the flow
loop, either independently of other additional containers, or in concert.

[0026]In all of the embodiments for solution exchange (solution mixing),
the liquid sample or samples in the at least one additional container,
when mixed with the liquid sample in the first container, can alter the
solution conditions of the first liquid sample in the first container, so
that the combined liquid is at a different solution condition that the
first liquid sample and/or the at least one additional liquid samples.
The solution conditions that can be changed include, but are not limited
to, pH, salt concentration, reducing agent concentration, oxidizing agent
concentration, both reducing agent concentration and oxidizing agent
concentration, chaotrope concentration, arginine concentration,
surfactant concentration, preferentially excluding compound
concentration, ligand concentration, concentration of any compounds
originally present in the solution, or addition of another reactant or
reagent.

[0027]In all of the foregoing embodiments of the devices, the device can
comprise a material that permits a change in oxygen concentration due to
oxygen mass transfer across the material of no more than about 0.2 mM in
the sample during the duration of the high pressure treatment. In another
embodiment, the material permits a change in oxygen concentration due to
oxygen mass transfer across the material of no more than about 0.1 mM in
the sample during the duration of the high pressure treatment. In another
embodiment, the material permits a change in oxygen concentration due to
oxygen mass transfer across the material of no more than about 0.05 mM in
the sample during the duration of the high pressure treatment. In another
embodiment, the material permits a change in oxygen concentration due to
oxygen mass transfer across the material of no more than about 0.025 mM
in the sample during the duration of the high pressure treatment. In
another embodiment, the material permits a change in oxygen concentration
due to oxygen mass transfer across the material of no more than about
0.01 mM in the sample during the duration of the high pressure treatment.
In another embodiment, the material permits a change in oxygen
concentration in the sample due to oxygen mass transfer across the
material of no more than about 10% of the initial oxygen content of the
sample during the duration of the high pressure treatment. In another
embodiment, the material permits a change in oxygen concentration in the
sample due to oxygen mass transfer across the material of no more than
about 5% of the initial oxygen content of the sample during the duration
of the high pressure treatment. In another embodiment, the material
permits a change in oxygen concentration in the sample due to oxygen mass
transfer across the material of no more than about 2.5% of the initial
oxygen content of the sample during the duration of the high pressure
treatment. In another embodiment, the material permits a change in oxygen
concentration in the sample due to oxygen mass transfer across the
material of no more than about 1% of the initial oxygen content of the
sample during the duration of the high pressure treatment. In the
foregoing embodiments, the high pressure treatment can last for about 6
hours, about 12 hours, about 18 hours, about 24 hours, about 30 hours,
about 36 hours, about 42 hours, or about 48 hours.

[0028]In another embodiment, the invention embraces methods of altering
solution conditions under high pressure, comprising the steps of:
providing at least one composition in a solution in a first container;
providing at least one agent for changing solution conditions in at least
one additional container, where the contents of the at least one
additional container are not in contact with the contents of the first
container; placing the containers under high pressure; and causing the
contents of the at least one additional container to contact the contents
of the first container. In another embodiment, the contents of the first
and at least one additional container are mixed by convection. In another
embodiment, the contents of the first and at least one additional
container are mixed by agitation. In another embodiment, the contents of
the first and at least one additional container are mixed by diffusion.
In another embodiment, the contents of the first and at least one
additional container are mixed by passing the contents through a mixer,
such as a static mixer. In another embodiment, the contents of the first
container and the at least one additional container are transferred to a
receiving container, where the receiving container may be empty prior to
transfer or may contain a liquid or solid composition prior to transfer;
the contents of the first container and the at least one additional
container can be mixed during or after the transfer to the receiving
container. In another embodiment, the at least one additional container
is contained within said first container. In another embodiment, the at
least one additional container is in a flow path with the first
container.

[0029]In one embodiment, the invention embraces methods of altering
solution conditions under high pressure, comprising the steps of:
providing at least one composition in a solution in a first container;
providing at least one agent for changing solution conditions in at least
one additional container, where the contents of the at least one
additional container are not in contact with the contents of the first
container; placing the containers under high pressure; and causing the
contents of the at least one additional container to contact the contents
of the first container, wherein the contents of the at least one
additional container are caused to contact the contents of the first
container over a period of time. In one embodiment, the contents of the
at least one additional container are caused to contact the contents of
the first container in a continuous manner, whereby the solution
conditions of the contents of the first container are changed
continuously over a period of time. In another embodiment, the contents
of the at least one additional container are caused to contact the
contents of the first container in a step-wise (discontinuous) manner
(e.g., by mixing portions of solutions, waiting, and mixing additional
portions of solutions), whereby the solution conditions of the contents
of the first container are changed step-wise over a period of time. In
one embodiment of this step-wise change in solution conditions, the pH of
the contents of the first container is at about 9 to about 11, or at
about 9.5 to about 10.5, or at about 10. In another embodiment of this
step-wise change in solution conditions, the pH of the contents of the
first container is at about 9 to about 11, or at about 9.5 to about 10.5,
or at about 10, and is lowered to a pH of about 7 to about 8.9, or about
7.5 to about 8.5, or about 8. In another embodiment of the stepwise
method, the pH is lowered by about 0.01 to about 2 pH units every
approximately 24 hours, or by about 0.1 to about 1 pH unit every
approximately 24 hours, or by about 0.1 to about 0.5 pH units every
approximately 24 hours, or by about 0.1 to about 0.4 pH units every
approximately 24 hours, or by about 0.1 to about 0.3 pH units every
approximately 24 hours, or by about 0.2 pH units every approximately 24
hours.

[0030]In one embodiment of the method, the at least one composition in a
solution in a first container is a protein. The protein can be in a
non-native state, such as a denatured protein or an aggregated protein;
the aggregated protein can be a soluble aggregate, insoluble aggregate,
or inclusion body, or any mixture of the forgoing.

[0031]In one embodiment of the method, the at least one agent for changing
solution conditions is an agent for changing the pH of the solution. In
another embodiment, the at least one agent for changing solution
conditions is an agent for changing the salt concentration of the
solution. In another embodiment, the at least one agent for changing
solution conditions is an agent for changing the reducing agent
concentration, oxidizing agent concentration, or both reducing agent
concentration and oxidizing agent concentration of the solution. In
another embodiment, the at least one agent for changing solution
conditions is an agent for changing the chaotrope concentration of the
solution. In another embodiment, the at least one agent for changing
solution conditions is an agent for changing the concentration of
arginine of the solution. In another embodiment, the at least one agent
for changing solution conditions is an agent for changing the
concentration of surfactant of the solution. In another embodiment, the
at least one agent for changing solution conditions is an agent for
changing the preferentially excluding compound concentration of the
solution. In another embodiment, the at least one agent for changing
solution conditions is an agent for changing the ligand concentration of
the solution. In another embodiment, the at least one agent for changing
solution conditions is an agent for changing the concentration of any
compounds originally present in the solution. In another embodiment, the
at least one agent for changing solution conditions is an additional
reactant or reagent to add to the solution.

[0032]In another embodiment, the containers are placed under at least
about 250 bar of pressure. In another embodiment, the containers are
placed under at least about 400 bar of pressure. In another embodiment,
the containers are placed under at least about 500 bar of pressure. In
another embodiment, the containers are placed under at least about 1000
bar of pressure. In another embodiment, the containers are placed under
at least about 2000 bar of pressure. In another embodiment, the
containers are placed under at least about 2500 bar of pressure. In
another embodiment, the containers are placed under at least about 3000
bar of pressure. In another embodiment, the containers are placed under
at least about 4000 bar of pressure. In another embodiment, the
containers are placed under at least about 5000 bar of pressure. In
another embodiment, the containers are placed under at least about 6000
bar of pressure. In another embodiment, the containers are placed under
at least about 7000 bar of pressure. In another embodiment, the
containers are placed under at least about 8000 bar of pressure. In
another embodiment, the containers are placed under at least about 9000
bar of pressure. In another embodiment, the containers are placed under
at least about 10,000 bar of pressure.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033]FIG. 1 depicts a top view of one embodiment of the invention, the
multi-well plate design.

[0034]FIG. 2A depicts a side view of one possible embodiment of the
multi-well design. The tops of the wells are partially covered in a
"dome" to ensure venting of all air.

[0035]FIG. 2B depicts a side view of the "dome" covering the wells in FIG.
2A.

[0036]FIG. 3 depicts an example of the 96-well plate embodiment with
sealing mat and clamp assembly that can be used to seal the "dome" inlets
of FIG. 2A and FIG. 2B.

[0037]FIG. 4 depicts another embodiment of the invention, where
heat-sealed septums are used to seal the wells of a multi-well embodiment
of the invention.

[0038]FIG. 5 depicts another embodiment of the invention, the constant
loading volume container.

[0039]FIG. 6 depicts another embodiment of the invention, the variable
loading volume container.

[0040]FIG. 7A depicts a sectional view of a one-way valve assembly for use
in the variable loading volume container. FIG. 7B depicts the one-way
valve assembly as installed in the variable loading volume container.

[0041]FIG. 8 depicts an embodiment of the invention useful for mixing
solutions at high pressure. In FIG. 8A, the secondary container is
depicted in closed position. In FIG. 8B, the secondary container is
depicted in open position.

[0042]FIG. 9 depicts another embodiment of the invention useful for mixing
solutions at high pressure.

[0043]FIG. 10 depicts results of an experiment demonstrating oxygen
transfer through materials which are not substantially oxygen-impermeable
at high pressure. The effects of storage and pressurization conditions on
oxygen transfer and GSH concentration are shown; the solutions conditions
were pH 8.0, 4 mM GSH, 2 mM GSSG, 500 ml solution, 25° C., for 17
hours.

[0044]FIG. 11 depicts the calculated transfer of oxygen through the walls
of a syringe made from various polymers (LDPE, low density polyethylene,
top curve; HDPE, high density polyethylene, second curve from top; PS,
polystyrene, third curve from top and second curve from bottom; PET,
polyethylene-terephthalate, bottom curve), as a function of the oxygen
concentration of the surroundings. Oxygen transfer is calculated for
syringe walls as a function of polymer type, assuming 24 hours for
transfer, 1/16 inch thickness, 1.5 inches length, and 0.25 inch outer
diameter.

[0045]FIG. 12 depicts the amount of oxygen loaded in a sample containing
an air bubble as a function of the bubble size in the sample, where the
bubble size is calculated as the volume percent of the sample. The curve
assumes PV=nRT, which is a suitable approximation for this calculation.

[0046]FIG. 13 depicts an overall view of an embodiment of a solution
exchange device.

[0047]FIG. 14 depicts a cross-section of the solution exchange device of
FIG. 13.

[0048]FIG. 15 depicts the pressure chamber portion of the solution
exchange device of FIG. 13 and FIG. 14, prior to mixing of solutions.

[0049]FIG. 16 depicts the pressure chamber portion of the solution
exchange device of FIG. 13 and FIG. 14, subsequent to mixing of
solutions.

[0050]FIG. 17 depicts one of the pre-mixing containers of the solution
exchange device of FIG. 13 and FIG. 14.

[0055]FIG. 21 depicts a variable loading volume embodiment of the
invention, with the check valve adapter (with check valve installed, as
depicted in FIG. 20A) inserted into the device to contain the liquid
therein.

[0056]FIG. 22 depicts an experiment performing Coomassie Blue solution
exchange under pressure. The open squares represent the actual sample
(upper right square lying on solid line corresponds to initial
conditions; lower right square with error bars corresponds to conditions
after solution exchange). The solid line represents the calibration line
from known concentrations of dye.

[0058]By "high pressure" is meant a pressure of at least about 250 bar.
The pressure at which the devices of the invention are used can be at
least about 250 bar of pressure, at least about 400 bar of pressure, at
least about 500 bar of pressure, at least about 1 kbar of pressure, at
least about 2 kbar of pressure, at least about 3 kbar of pressure, at
least about 5 kbar of pressure, at least about 6 kbar of pressure, at
least about 7 kbar of pressure, at least about 8 kbar of pressure, at
least about 9 kbar of pressure, or at least about 10 kbar of pressure.

[0059]By "closed system" is meant the standard chemical thermodynamic term
referring to a system where matter cannot be transferred between the
system and its surroundings; however, transfer of mechanical or heat
energy can occur between a closed system and its surroundings. In
contrast, an "open system" permits transfer of matter and/or mechanical
or heat energy between the system and its surroundings. An "isolated
system" is a closed system that does not permit either mechanical or
thermal contact with its surroundings, i.e., no transfer of mechanical or
heat energy takes place to or from an isolated system. A "substantially
closed system" is a system where less than about 1%, more preferably less
than about 0.5%, more preferably less than about 0.2%, more preferably
less than about 0.1%, more preferably less than about 0.05%, still more
preferably less than about 0.01% of the mass of the sample can be
transferred between the system and its surroundings.

[0060]By "significant transfer of liquid sample" is meant a transfer of
about 1% or more of the volume of liquid contained in a sample (measured
at standard atmospheric pressure). When devices of the invention are
designed to prevent significant transfer of liquid sample, the amount of
sample transferred during the use of the device is less than about 1%,
more preferably less than about 0.5%, more preferably less than about
0.2%, more preferably less than about 0.1%, more preferably less than
about 0.05%, still more preferably less than about 0.01% of the
unpressurized volume of the sample.

[0061]By "substantially impermeable to oxygen at high pressure,"
"substantially oxygen-impermeable at high pressure," or "substantially
impermeable to oxygen mass transfer at high pressure" is meant a material
that permits a change in oxygen concentration due to oxygen mass transfer
across the material of no more than about 0.3 mM in the sample during the
duration of the high pressure treatment. In another embodiment, the
material permits a change in oxygen concentration due to oxygen mass
transfer across the material of not more than about 0.2 mM, preferably
not more than about 0.1 mM, preferably not more than about 0.05 mM, more
preferably not more than about 0.025 mM, still more preferably not more
than about 0.01 mM, in the sample during the duration of the high
pressure treatment. In percentage terms, the material permits a change in
oxygen concentration in the sample due to oxygen mass transfer across the
material of no more than about 10%, preferably no more than about 5%,
more preferably no more than about 2.5%, still more preferably no more
than about 1%, of the initial oxygen content of the sample during the
duration of the high pressure treatment.

Materials for High-Pressure Devices

[0062]The body of the high-pressure devices can be fabricated from a wide
variety of materials. If a device does not have at least one moveable
surface which can transmit pressure (such as the variable loading volume
device depicted in FIG. 6), then the materials from which the device is
made should be flexible to enable pressure transfer. Suitable materials
should not break, fracture, or otherwise undergo any failure or loss of
integrity under high pressure treatment which would permit leakage of
samples either from one or more sample compartments to the external
surroundings, or allow leakage of fluids, gases, or other materials in
the external surroundings of the container into the one or more sample
compartments, or which would permit leakage of samples between sample
compartments. Such leakage, of course, is not meant to include
intentional transfers between one or more sample compartments and the
external surroundings, or intentional transfers between two or more
sample compartments or other compartments, which are deliberately desired
by the artisan.

[0063]The device must be constructed with a material that can withstand at
least about 250 bar of pressure and still maintain integrity. In another
embodiment, the material can withstand at least about 500 bar of pressure
and still maintain integrity. In another embodiment, the material can
withstand at least about 1 kbar of pressure and still maintain integrity.
In another embodiment, the material can withstand at least about 2 kbar
of pressure and still maintain integrity. In another embodiment, the
material can withstand at least about 3 kbar of pressure and still
maintain integrity. In another embodiment, the material can withstand up
to about 5 kbar, preferably up to about 10 kbar of pressure, and still
maintain integrity. The specified pressures are multi-dimensional
pressure on the device, not a pressure differential or pressure drop
across the device. That is, the container or containers used as the
device are placed in a pressure chamber which is pressurized to the
specified pressure; while the pressure chamber must be capable of
withstanding a pressure differential or pressure drop of up to 5 kbar or
10 kbar within the chamber versus atmospheric pressure outside of the
chamber, the material of the container does not experience such a
dramatic differential pressure, but rather a uniform pressure from all
directions.

[0064]The material used should also permit pressure transfer from the
surroundings to the sample compartments, so that the pressure across the
device is roughly equivalent; that is, the difference in pressure
experienced by any two locations within the device is no more than about
1%, preferably no more than about 0.5%, more preferably no more than
about 0.1%, of the total pressure. In other embodiments, the absolute
difference in pressure experienced by any two locations within the device
is less than about 5 bar, preferably less than about 2 bar, more
preferably less than about 1 bar. Any difference between the external
applied pressure and any interior portion of the device is no more than
about 5%, preferably no more than about 2%, more preferably no more than
about 1%, still more preferably no more than about 0.5%, yet more
preferably no more than about 0.1%, of the total applied external
pressure. Therefore, the material should be flexible in order to transmit
pressure.

[0065]In one embodiment, the materials are polymers. In another
embodiment, the polymeric materials can be injection molded for
inexpensive mass production. Suitable polymeric materials include
polyethyleneterephthalate, high-density polyethylene, polystyrene, and
polystyrene-butadiene block copolymers. Other polymeric materials which
can withstand high-pressure treatment, but which are not necessarily
oxygen impermeable, include low-density polyethylene, polypropylene, and
polycarbonate.

Considerations of Oxygen Content of Sample

[0066]Many reactions, such as refolding of cysteine-containing proteins,
can be affected by the oxygen content of the sample. Typically a protein
refolding experiment will entail use of a specified concentration of
redox reagents such as thiols (e.g., glutathione, cystamine, cystine,
dithiothreitol, dithioerythritol; in reduced form, oxidized form, or a
mixture of reduced and oxidized forms for, e.g., disulfide shuffling).
The concentration of oxygen in a sample can be affected by the presence
of air bubbles in a sample, as air bubbles will be forced into solution
at high pressures, changing the O2 concentration in the sample. The
concentration of oxygen in a sample can also be affected by diffusion of
oxygen across the walls of the device. The sample device will typically
be placed in a chamber to which pressure is applied; if the fluid used in
the chamber is water, then oxygen dissolved in the chamber's water
surrounding the sample device can diffuse across the walls of the device.

[0067]These considerations are addressed in the following sections,
"oxygen concentration changes due to air bubbles," and "oxygen
permeability at high pressure."

Oxygen Concentration Changes Due to Air Bubbles

[0068]It is estimated that about 80% of the variation in oxygen
concentration will arise from air bubbles in the sample, while about 20%
of the variation will arise from oxygen diffusion across the walls of a
syringe-type device (where the device is not substantially impermeable to
oxygen transfer at high pressure). This underscores the importance of
removing as many air bubbles as possible from the sample vial. For every
25 μl of air in a 1 ml sample, 0.2 mmoles O2 is loaded, as high
pressure will dissolve the air into the liquid sample; that amount of
oxygen will react with 0.8 mM reduced thiol. Typical reduced thiol
concentrations range from about 1 mM to about 10 mM (Clark E. D.,
"Protein refolding for industrial processes," Curr. Opin. Biotechnol.
12:202-207 (2001)), and over this range a 0.8 mM change in reduced thiol
concentration will cause a variation in concentration of from about 8% to
about 80%. At a typical concentration of 4 mM reduced thiol, a 0.8 mM
reduction of reduced thiol results in about a 20% change in solution
concentration of reduced thiols. This underscores the importance of
removing all air bubbles, which is difficult to accomplish with current
state-of-the-art vials and which the instant invention is designed to
address.

[0069]FIG. 12 shows the oxygen loading caused by air bubbles of various
sizes. The volume percent of air bubbles should be kept as low as
possible, to no more than about 10% of the sample volume, more preferably
no more than about 5% of the sample volume, still more preferably no more
than about 2.5% of the sample volume, yet more preferably no more than
about 1% of the sample volume.

Oxygen Permeability at High Pressure

[0070]The materials used in the devices are optionally substantially
impermeable to oxygen mass transfer at high pressure. Materials which are
substantially impermeable to oxygen should be used when oxygen transfer
may affect the sample being studied or treated using the device.
Optionally, the materials used are also substantially impermeable to
transfer of other gases at high pressure, such as carbon dioxide, which
may affect the sample being studied or treated using the device.
Materials which are substantially impermeable to oxygen mass transfer at
high pressure include, but are not limited to, polyethylene-terephthalate
(PET or PETE), Mylar® (Mylar is a registered trademark of DuPont,
designating a biaxially-oriented polyethylene terephthalate polyester
film), high-density polyethylene, and polystyrene. Alternatively, if
vessels walls are made thick enough, materials which are less impermeable
to oxygen can be used. Finally, materials which are more permeable to
oxygen, including, but not limited to, polystyrene-butadiene block
copolymers such as Styrolux® (e.g., Styrolux® 684D) can be used
with suitable coatings of other polymers or other materials to decrease
their oxygen permeability. (Styrolux® is a registered trademark of
BASF Aktiengesellschaft Corp., Ludwigshafen, Germany, and Westlake
Plastics Company, Lenni, Pa., for styrene resins.)

[0071]Experimental evidence confirms the utility of using substantially
oxygen-impermeable materials at high pressure when oxygen affects the
sample being studied or treated using the device. Up to about 0.35
micromoles of O2 can be transferred during a typical pressure
experiment, enough to significantly alter the redox environment of a
solution. FIG. 10 depicts an experiment done with conventional syringes
currently used for high-pressure treatment. The syringes used were 1 ml
low-density polyethylene syringes from Becton Dickinson. A 500 ml aqueous
solution at pH 8.0, 4 mM GSH (reduced glutathione), 2 mM GSSG (oxidized
glutathione), was kept at 2150 bar for 17 hours. As indicated in FIG. 10,
enough oxygen was transferred to lower the concentration of reduced
glutathione from 4.0 mM to 3.5 mM or less.

[0072]FIG. 11 depicts calculations of the amount of oxygen transfer across
the walls of a syringe used under high pressure. The calculations are for
a syringe of 1/16 inch thickness, 1.5 inch length, and 0.25 inch outer
diameter. The calculation assumed a 24 hour experiment at 2000 bar with a
variable surrounding oxygen concentration; the expected oxygen
concentration in the surrounding fluid will likely be about 0.3 mM (under
the assumption that about 10% of the volume of the surroundings is made
up of an air bubble before compression), and is indicated with a vertical
dashed line in FIG. 11. The oxygen in the bubble is calculated by simply
using the ideal gas law to calculate the amount of air in the bubble at
standard temperature and pressure; at higher pressure, the air will
dissolve into the solution. The calculation is performed using Fick's law
of diffusion at steady-state; diffusion coefficients are used instead of
permeability coefficients, as the solubility of O2 in polymers
increases dramatically at high pressures. The diffusion coefficients were
taken from the Polymer Handbook, 4th Edition; editors, J. Brandup,
E. H. Immergut, and E. A. Grulke; associate editors, A. Abe, D. R. Bloch;
New York: Wiley, 1999.

[0073]With these assumptions, the calculations indicate that, at the
likely value of oxygen concentration in the surrounding liquid,
approximately 0.2 mM equivalents of O2 is transferred in tubes made
of HDPE, and 0.6 mM equivalents of O2 with LDPE. Oxygen transfer
across a polypropylene device was not calculated, but based on relative
permeability values, is believed to lie between the values for HDPE and
LDPE. Polyethylene terephthalate (PET or PETE) is calculated to have
almost no transfer of oxygen under the conditions assumed. Consequently,
this calculation demonstrates that materials can be judiciously chosen to
significantly reduce or almost eliminate oxygen transfer through the
polymeric walls of the devices.

Device Embodiments: Multi-Well Plates

[0074]In one embodiment, the high-pressure device comprises a plurality of
wells in a body or plate ("multi-well plate"). One example of such an
embodiment is shown in FIG. 1. The embodiment shown is a 96-well plate; a
body (1) made of a flexible material substantially impermeable to oxygen
mass transfer at high pressure has ninety-six wells (2) for holding
liquid samples. The material is preferably (but not necessarily) chosen
so that the plate can be formed by injection molding.

[0075]Once a suitable material has been chosen for the body of the
multi-well plate embodiment, the samples must be introduced into the
sample compartments. The inclusion of air pockets in the sample wells is
undesirable, as the oxygen in the air will be driven into solution under
high pressure, altering the redox environment of the sample, and the
presence of air pockets may also cause excessive strain on the material.
The wells are thus designed so as to eliminate, to the greatest extent
possible, any residual air left in the wells.

[0076]FIG. 2A depicts a side view of one possible embodiment of the well
design (1). The wells (3) are partially covered in a "dome" (4) to ensure
venting of all air. The dome (4) is shown in larger detail in FIG. 2B.
The region (6) is the inlet for sample loading, and is surrounded by
solid material (5) forming the dome. The inlet (6) should be large enough
to enable insertion of the appropriate sized pipette tip for reagent
delivery and the venting of air. This domed design enables overfilling to
vent all air in the well prior to sealing. Additionally, during the
overfill, excess sample will drain down the sides of the dome and will
eliminate cross-contamination between samples. The dome (4) has a
substantially flat surface on top, in an (4A) area closely surrounding
the inlet, in order to provide an adequate sealing surface. The
dimensions are selected to enable sample loading with standard-sized
pipette tips, to enable sample venting, to have sufficient troughs at the
base of the domes to prevent cross-contamination, and to provide the
previously mentioned flat top to provide an adequate sealing surface.

[0077]In one embodiment of the multi-well device, a mat is placed on the
top of the multi-well plate to seal the wells. Materials suitable for
such a mat include, but are not limited to, silicone rubber. In one
embodiment, the mat has a thickness of approximately 1/8 inch, with
length and width substantially identical to that of the multi-well plate
it is to be used with. The mat should be made from a material of
sufficient flexibility to enable a good seal on top of the domes, and to
allow deformation due to pressure-induced volumetric changes within the
sample. As there is no differential pressure across the sealing surface
in this embodiment--that is, the pressure experienced by the sample
inside the well is substantially similar to the pressure experienced by
the mat--the sealing mat need not provide any additional sealing capacity
than that expected at atmospheric pressure. If the sealing material used
is not substantially impermeable to oxygen at high pressure, a film which
is substantially impermeable to oxygen at high pressure can be placed on
the sealing mat to inhibit oxygen transfer. The film can be made from
materials including, but not limited to, Mylar®. In this embodiment
of the multi-well device using a sealing mat, a clamp is affixed on the
plate in order to place force on the sealing mat and enable sealing of
the wells. The clamp should provide uniform force across the device, and
sufficient force to ensure an adequate seal. The clamp should also
provide constant force throughout the pressurization cycle, which
requires a constant tension clamp (not a constant force clamp) due to the
contraction of materials (especially the sealing mat) at high pressure.
FIG. 3 depicts the 96-well plate embodiment with sealing mat (3-1) and
clamp assembly (3-2).

[0078]In another embodiment of the multi-well plate, depicted in FIG. 4,
the wells of the plate are not covered by a dome and sealing mat;
instead, the wells are covered by heat-sealed septa (4-2) prior to
loading the wells with samples. Such septums are commonly used when
sealing medical vials. The heat sealed septum ensures a sealed well and
prevents sample contamination. Samples are loaded into this embodiment of
the multi-well plate by injection with a multi-channel pipetter equipped
with needles rather than pipettes. The needles penetrate the septum in
order to fill the sample wells. A secondary needle also pierces the
sample concurrently with filling, in order to vent air and to allow the
well to fill completely. Multi-channel pipetters are available
commercially which are designed for pipetting solutions into multi-well
plates; such a pipetter can be easily adapted to use a needle for sample
loading instead of a pipette tip. After sample loading, the septum is
covered with a secondary, adhesive polymeric membrane (4-1). The membrane
seals the pierced holes created during sample loading. Optionally, the
membrane can also inhibit oxygen diffusion across the septum; that is,
the membrane can be substantially impermeable to oxygen. Potential
materials for the adhesive polymeric membrane include, but are not
limited to, Mylar®.

Device Embodiments: Constant Loading Volume Devices

[0079]In another embodiment, the high-pressure device comprises a
container where the volume of the container is fixed at standard
pressure; this embodiment is designated the constant loading volume
device. The entire container shrinks proportionally upon exposure to high
pressure; typically, the container will shrink by about 5%-10% of its
volume at 2 kbar, and by about 20% (estimated) of its volume at 4 kbar;
hence, for fabricating this device, a flexible material should be used.
One example of such an embodiment is shown in FIG. 5. This device
consists of a cylindrical barrel which has a conical bottom. The
container can be fabricated with a wide variety of internal volumes;
examples of dimensions for containers having 250 μL, 500 μL, 750
μL, or 1000 μL are specified in Table 1. The interior of the
container can be graduated, for example at 50 μL increments. The top
of the cylindrical barrel can be threaded for seal with a screw cap
(which can be shaped as the conical bottom in FIG. 5, or which can also
be cylindrical). The threaded screw cap should be capable of maintaining
a seal when there is at least about 5 psig pressure differential between
the interior and exterior (note that the 5 psig is a differential
pressure, not a total pressure; pressure differentials of this magnitude
are similar to those of commercial bottles containing carbonated
beverages). Typical sizes of embodiments of the constant loading volume
device are given in Table 1 (the thickness of the walls of these
particular embodiments of the constant loading volume device is 1/16
inch).

[0080]In another embodiment, the high-pressure device comprises a
container of variable loading volume. An example of this embodiment is
shown in FIG. 6. In this embodiment, a cylinder (6-1) with a moveable
plug (6-2) and a removable cap (6-3) is provided, in a fashion similar to
a syringe. In one embodiment, when the cylinder is vertically oriented,
the plug forms the bottom of the sample compartment, and acts as a seal
between the sample compartment and the external environment. The plug can
have an attached plunger rod, or a separate plunger rod (6-4), which can
be used to move the plug to the desired volume before filling the
cylinder. The cylinder can be graduated, for example in 50 μL
increments, in order to guide placement of the plug to the desired
volume. The sample is contained in the interior space (6-5) of the
device. The cylinder can then be filled with the desired sample, with
care taken to remove as much residual air as possible. The removable cap
is then placed on the top of the cylinder. In an alternative embodiment,
the cylinder can be filled in the opposite orientation, i.e., the
cylinder can be oriented so the cap is placed on the bottom of the
cylinder, and the plug is inserted into the top of the cylinder. In one
embodiment, the removable cap is threaded, and can simply be screwed on
to the top of the cylinder. The threaded removable cap should be capable
of maintaining a seal when there is at least about 5 psig pressure
differential between the interior and exterior (note that the 5 psig is a
differential pressure, not a total pressure; pressure differentials of
this magnitude are similar to those of commercial bottles containing
carbonated beverages). The sample can then be treated under pressure;
after the pressure treatment, the cap can be removed and the contents of
the cylinder are poured out, or pushed out by pushing the plug with the
plunger rod.

[0081]In an alternative embodiment, the removable cap is replaced with a
breakable tip on the closed end of the cylinder. In this embodiment, a
liquid sample is placed in the cylinder and the plug is inserted at the
top of the cylinder. The breakable tip is kept intact during the
high-pressure treatment. After the treatment, the tip is broken off, and
the contents of the cylinder are poured out, or pushed out by pushing the
moveable plug with the plunger rod. The tip can be designed to be broken
off by hand, or can be designed to be broken off by a cutting tool; see
FIG. 21 for an example of a variable loading volume device where the tip
can be removed by a cutting tool in order to expel the sample.

[0082]In another alternative embodiment, a needle can be run between the
moveable plug and the cylinder wall to insert the sample into the
cylinder. A one-way valve on the moveable plug allows expulsion of air in
the cylinder as the sample is introduced (see FIG. 7A, FIG. 7B, and
discussion of a one-way valve assembly below).

[0083]It should be noted that, as the moveable plug will move in response
to applied pressure, the material used to fabricate the variable loading
volume device need not be as flexible as the material used in the other
devices of the invention.

[0084]In FIG. 7A and FIG. 7B, a moveable plug (7) is shown which is
particularly adapted for high-pressure applications, and which can
function as a one-way valve plug. The embodiment shown in FIG. 7A and
FIG. 7B is a flap plug or flap valve, as it relies on a flap to allow
one-way flow of liquid. In FIG. 7A flexible flap (7-1) forms a seal with
the O-ring (7-2). The flap (7-1) and O-ring (7-2) seal the external
environment (7-6) from the internal passage (7-4), which opens to the
interior of the variable loading volume device at opening (7-7). Area
(7-3) is solid. When the one-way valve plug is inserted into the
polymeric barrel (6-1) of the variable loading volume device as depicted
in FIG. 7B, the plug can be pressed down until sample begins to bleed out
of the one-way valve. This occurs as the flexible flap (molded flap)
bends to allow sample to escape via the path indicated by the arrows in
FIG. 7B. Pressing down until sample bleeds out of the valve ensures
exclusion of as much air as possible from the sample, and also allows
adjustment of the amount of sample in the device. As the flap can bend
only in one direction (away from the O-ring), neither air nor any other
substance present external to the sample can flow back into the sample.

[0085]Another variable loading volume device suitable for use as a high
pressure sample vial is shown in FIG. 21, comprised of a polymeric sample
barrel, a check valve adaptor, a check valve assembly, and an O-ring
seal. In FIG. 19, another design for a moveable plug (19-0) is shown
which is particularly useful for high-pressure applications, and which
can function as a one-way valve plug; this moveable plug also functions
as a check valve adapter (that is, a check valve can be inserted into the
moveable plug (19-0)). The plug can be manufactured from a variety of
materials, including, but not limited to, Delrin® (Delrin® is a
registered trademark of E. I. Du Pont de Nemours and Company, Wilmington,
Del., for acetal resin); the plug can be fabricated by injection molding
or by machining. The area of the plug that contacts the liquid sample is
curved (19-1) (note that the plug would be inverted from the orientation
shown in FIG. 19 when inserted into a container holding a liquid); this
curvature ensures that as much air as possible is forced out of the
container. Indentation (19-3) allows installation of an O-ring to form a
moveable seal with the walls of the container. This plug is adapted to
receive a check valve in its interior lumen (19-4), which can be easily
installed by manually inserting the check valve into the adapter. The
valve plug/check valve adapter preferably utilizes a ball-and-spring
check valve. FIG. 20 depicts such a check valve (20-1), which is
commercially available (The Lee Co, PN#CCPX0003349S A, Westbrook, Conn.).
The arrow in FIG. 20 indicates the direction of permitted liquid flow in
the check valve. Liquid passes through lumen (20-2) of the check valve,
pushing the ball of the valve (20-3) down by compressing the valve spring
(20-4). Once fluid no longer flows, spring (20-4) pushes ball (20-3) back
to seal the valve. The check valve of FIG. 20 is inserted into the check
valve adapter component of FIG. 19; FIG. 20A depicts the check valve
adapter (19-0) with the check valve (20-1) installed. The check valve
adapter containing the check valve is inserted into a container to form a
variable loading volume container as in FIG. 21. The container (21-1) is
made of a flexible material which can withstand pressurization up to
about 5 kbar, preferably up to about 10 kbar and optionally is
substantially impermeable to oxygen. The container (21-1) of the variable
loading volume device can be injection molded in a single cavity mold
(such as those available from PTG Global Inc., Orange County, California)
using materials such as Styrolux® 684D. The material of construction
is not limited to Styrolux®, and could be further adjusted to
modulate oxygen permeability. The container contains liquid sample
(21-2). In one embodiment, the variable loading volume device can hold a
liquid volume of up to 1.2 mls, and can be used in the volume range of
150-1200 uL. Adapter (21-3) with check valve (21-4) is inserted into the
top of the container (21-1); the check valve is oriented so that air and
fluid in the container can flow up and out of the container, but fluid is
blocked from flowing down and into the container. As the adapter is
pushed down into the container, air is forced out of the check valve; the
concave bottom of the adapter ensures that as much air as possible is
forced out before liquid sample begins to be forced out of the container.
The variable loading volume device as depicted can then be subjected to
high pressure.

Device Embodiments. Solution Exchange (Solution Mixing) Devices

[0086]In another embodiment, the high-pressure device comprises a
plurality of compartments, where the contents of the compartments can be
kept separate or can be mixed together. Such devices are designated as
solution exchange or solution mixing devices. When treating a liquid
sample at high pressure, the contents of the containers can be mixed to
alter the chemical solution conditions of the liquid sample. The chemical
solution conditions which can be changed include, but are not limited to,
any one or more of pH, salt concentration, reducing agent concentration,
oxidizing agent concentration, chaotrope concentration, concentration of
arginine, concentration of surfactant, preferentially excluding compound
concentration, ligand concentration, the concentration of any compounds
originally present in the liquid sample, or addition of an additional
reactant or reagent to add to the solution. In another embodiment, the
chemical solution conditions are changed by adding an additional reagent
or reactant to the liquid sample. Such a reagent or reactant may comprise
an enzyme inhibitor, a drug, a small organic molecule (of molecular
weight below about 1000 Daltons), or a protein derivatization reagent.

[0087]Container(s)-in-container embodiment: In one such embodiment
comprising a plurality of compartments where the contents of the
compartments can be kept separate or can be mixed together, the
high-pressure device comprises a primary compartment enclosing one or
more secondary compartments, where the one or more secondary compartments
can be opened without opening the primary compartment, whereby the
contents of the one or more secondary compartments are released into
contact with the contents of the primary compartment. An example of this
embodiment is shown in FIG. 8A and FIG. 8B. A variable loading volume
container, such as the variable loading volume container of FIG. 6 or
FIG. 21, is used as the primary compartment, while one or more secondary
containers (8-1) are placed within the interior (6-5) of the variable
loading volume container. (It should be noted that the variable loading
volume container is used simply as an example; any of the other devices
of the invention, such as the constant loading volume container, can be
used as the primary compartment.) One end of the secondary container(s)
is sealed. A magnetic disk (8-3) is placed on the other end of the
secondary container(s), which will have an axle built which passes
through one wall or side of the secondary container(s), through the
center of the disk, and into the facing wall or side of the secondary
container(s). The disk should be designed with a tolerance so as to fit
as precisely as possible inside the secondary container. The disk is
designed to freely rotate on the axle, effectively opening and closing
the secondary container(s) in a manner analogous to a conventional
butterfly valve. A chamfer or beveled edge is used to enable free
rotation of the magnetic disk with as tight a tolerance as possible on
the rear of the disk. This design enables the magnetic disk to freely
rotate, while providing an effective seal when the disk is in the closed
position. When this design is used, it is preferable to maintain the
primary container in a position such that the secondary container or
containers are in a vertical position, in order for gravity to assist in
maintaining the magnetic disk in its closed position. The switch is
actuated by electric coils placed in a vertical and horizontal fashion
around the exterior of the high pressure vessel in which the primary
compartment (which contains the secondary compartment(s)) is placed. The
horizontal coils are essentially parallel to the magnetic disks within
the pressure vessel, in order to generate a magnetic field which
maintains the magnetic disk in the closed or sealed position. This is
depicted in FIG. 8A. As pressure vessels are commonly made of stainless
steel, the coils are designed with the appropriate number of loops, gauge
thickness, and current to enable the generation of a magnetic field
strong enough penetrate into the interior of the pressure vessel.
Alternatively, the pressure vessel could be made out of a material that
does not attenuate the magnetic field as much as steel or other such
ferromagnetic materials. Another arrangement of electric coils for
control of the magnetic disks involves placing coils around the sample
rack in a horizontal and vertical manner. In this design, the magnetic
field would not have to penetrate the steel walls of the pressure vessel;
however, the wires carrying the current would have to run into the
interior of the pressure vessel. This can be accomplished by fabricating
the base of a sealing plug of a conventional pressure vessel out of an
insulating ceramic, rather than steel.

[0088]When the magnetic disks are to be kept in the closed position, the
horizontal field is turned on and the horizontal field is turned off,
maintaining the magnetic disk in the horizontal position and sealing the
solution contents of the secondary container from those of the primary
container. To enable solution exchange, current in the horizontal and
vertical coils is manipulated in the appropriate manner (e.g., turning
off the current in the horizontal coils and turning on the current in the
vertical coils) to open the disk as depicted in FIG. 8B, allowing the
contents of the secondary container(s) (contained in interior space (8-2)
of the secondary container) to contact the contents of the primary
container (contained in interior space (6-5) of the primary container).
The disk can be employed to generate mixing action; current in the
horizontal and vertical coils is alternated, with alternating current, to
generate a rotating electromagnetic field and flip the magnetic disk.
This opens the contents of the secondary container to the primary
container, while the motion of the disk enables convection and solution
exchange. In a variation, the cap(s) on the secondary container(s) can be
controlled by drive shafts which enter the high pressure chamber through
appropriately sealed openings into the high pressure chamber, and which
also pass through the primary container through appropriate seals.

[0089]Flow-loop embodiment: In another such embodiment comprising a
plurality of compartments where the contents of the compartments can be
kept separate or can be mixed together, the high-pressure device
comprises at least two compartments connected by flow paths, where the
compartments and the flow paths form a closed circular loop with at least
one pump. An example of this embodiment is shown in FIG. 9. A liquid
sample is placed in the "dissociation" chamber (9-1), while a second
solution is placed in the "refolding" chamber (9-2). Additional
dissociation chambers, refolding chambers, and flow paths can be added as
desired. The device is then placed in the pressure chamber (not shown)
and pressurized. When mixing of the liquid sample with the second
solution is desired, a piston pump (9-5) is turned on, circulating the
liquids through the closed circular loop (9-3). The piston (9-7) can be
made of a magnetized material, enabling control of the pump rate by a
magnetic field. Microprocessor-controlled battery-powered coils can be
placed inside the pressure chamber, along with the chambers and flow
loop, in order to control the piston pump. (The microprocessor (9-8) and
battery (9-9) are preferably embedded in an epoxy block (9-4) to reduce
pressure transfer to the microprocessor itself.) Alternatively, the
arrangement of metal coils for control of the secondary compartment metal
disk in the primary container/secondary container device can be used to
control the piston. In yet another variation, the device can be
controlled by drive shafts which enter the high pressure chamber through
appropriately sealed openings into the high pressure chamber. One or more
check valves (9-6) ensure unidirectional flow. While the containers are
labeled "dissociation chamber" and "refolding chamber" for ease of
understanding of the figure, it will be appreciated that other chemical
and biochemical processes can take place in either or both chambers.

[0090]Pre-mix container(s)/receiving (post-mix) container embodiment: In
another such embodiment comprising a plurality of compartments where the
contents of the compartments can be kept separate or can be mixed
together, the high-pressure device comprises a system comprising at least
two containers holding liquid samples designated pre-mix containers. The
liquid samples usually differ in one or more conditions or compositions,
such as salt concentration, pH, etc. (The liquid samples can be the same
if desired.) The system also comprises at least one additional container
designated the receiving container or post-mix container, where the
receiving (post-mix) container can be empty prior to transfer or can
contain a liquid or solid composition prior to transfer. Such a system
(100) is depicted in FIG. 13, and in detailed cross-section in FIG. 14.
In FIG. 14, a pressure chamber (102) sealed by plug (112) supports two
pre-mix containers, (120) and (122), which contain separate liquid
samples. The pre-mix containers are depicted as roughly equal in size in
FIG. 14; however, the size of the containers can be varied relative to
each other, so that, for example, one pre-mix container could have twice
the volume as the other pre-mix container. Also, for simplicity, only two
pre-mix containers are depicted, but more pre-mix containers can be used
if desired. The pre-mix containers have mobile pistons (128); liquid
conduits (124) lead to a mixer (126). The mixer leads to receiving
(post-mix) container (130) containing piston (132), which is depicted as
flush against the top of the receiving container in FIG. 14. Valve (104),
pressure generator (108), and pressure line (110) communicate with the
inside (103) of the pressure chamber (102), and can pressurize the inside
of the pressure chamber (102) up to, for example, 2,000-2,500 bar. Valve
(106), pressure generator (108), and hydraulic line (111) communicate
with liquid disposed beneath the piston (132). FIG. 15 shows the pressure
chamber (102) in more detail. Hydraulic outlet (134) removes liquid from
receiving container (130), causing piston (132) to be pulled away from
seal (131), i.e., piston (132) is drawn away from the inlet from mixer
(126). This then draws the liquids in pre-mix containers (120) and (122)
through mixer (126), where the liquids mix en route to receiving
container (130). Pistons (128) are pulled down as liquid exits containers
(120) and (122); when pre-mix containers (120) and (122) are emptied, the
pistons (128) rest against seals (125). In another embodiment (not
shown), hydraulic pressure can be applied to the external side of pistons
(128) to facilitate fluid expulsion from pre-mix containers (120) and
(122). FIG. 16 shows the apparatus after the liquid samples in pre-mix
containers (120) and (122) has been transferred to receiving container
(130). Pistons (128) are flush against seals (125) after fluid expulsion
from pre-mix containers (120) and (122). Piston (132) in receiving
container (130) has been pushed away from seal (131) to accommodate
liquid being transferred to the receiving container. FIG. 17 depicts
pre-mix container (120) in more detail. Sample is introduced into the
pre-mix container (120) through inlet (121); after introduction of
sample, a plug or check valve can then be inserted into inlet (121) to
seal the pre-mix container. Piston (128) has an annular indentation
(129A) where O-ring (129B) is seated in order to form a seal between the
piston and the wall of the container. FIG. 18 depicts the receiving
container (130) in more detail. Liquid enters the receiving container
through opening (136) in seal (131) (a check valve, not shown can
optionally be disposed in opening (136) in order to prevent backflow); an
O-ring (135) enhances the seal. Negative hydraulic pressure is applied
via opening (134), which pulls piston (132) downwards, which in turn
draws the liquid from the pre-mix containers (not shown in FIG. 18) into
the receiving container (130). Piston (132) has an O-ring (135) to
prevent fluid transfer around the piston.

[0091]Pressure chamber (102) can be pressurized so as to generate
2000-2500 bar (higher or lower values, such as 250 bar to 10 kbar, or 1
kbar to 10 kbar, or 1 kbar to 5 kbar, can also be employed) on the liquid
samples. Thus the pre-mix containers, the receiving container, and
consequently the liquid samples themselves can be maintained at high
pressure before, during, and after mixing. The device thus allows for two
or more solutions to be treated or incubated separately at high pressure
for a first period of time (for example, from about 1 minute to about 1
week, or about 10 minutes to about 48 hours, or about 1 hour to about 48
hours, or about 10 minutes to about 24 hours, or about 1 hours to about
24 hours, or about 10 minutes to about 12 hours, or about 1 hour to about
12 hours, or about 1 hour to about 6 hours). The solutions can then be
mixed together; the mixed solutions can be incubated for a second period
of time (for example, from about 1 minute to about 1 week, or about 10
minutes to about 48 hours, or about 1 hour to about 48 hours, or about 10
minutes to about 24 hours, or about 1 hours to about 24 hours, or about
10 minutes to about 12 hours, or about 1 hour to about 12 hours, or about
1 hour to about 6 hours). After both incubation periods are complete, the
pressure chamber is depressurized, and the solution is removed from the
receiving chamber, where it can be analyzed for various properties (such
as proper refolding of a protein) and/or used for a desired purpose.

[0092]Examples of equipment that can be used include: high pressure
generator, PN# 37-5.75-60, High Pressure Equipment Co., Erie, Pa. (in the
form of a syringe pump, rated to 60,000 psi); high pressure tubing (PN#
60-9H4-304, High Pressure Equipment Co.); high pressure valves (PN#
60-11HF4, High Pressure Equipment Co.); high pressure glands (PN#
60-2HM4) and collars (PN# 60-2H4 from High Pressure Equipment Co.). The
pre-mix containers can be manufactured from quartz Suprasil cylinders
(Wilmad Glass, Buena, N.J.); the quartz cylinders can be capped with
manufactured stainless steel pistons (High Precision Devices, Boulder,
Colo.) which are equipped with O-rings (McMaster-Carr, Aurora, Ohio, PN
9396K16, 2-011, made from silicon rubber) The outlet of the primary
chambers is connected to the static mixer through the use of standard
HPLC chromatography fittings (PN# F-300-01, F-113, F-126x, 1576,
Upchurch, Oak Harbor, Wash.). The mixing device as depicted in the
Figures is optional; when a mixing rate higher than simple diffusion is
desired, or when thorough mixing is desired, such a mixer can be
employed. Static mixers, such as those used in HPLC applications, can be
used; these can be obtained from numerous suppliers (for example,
Analytical Scientific Instruments, El Sobrante, Calif., static mixer PN#
40200000.5). The outlet of static mixer is connected to the secondary
refolding chamber through the use of standard HPLC chromatography
fittings (e.g., the Upchurch fittings as previously described).

[0093]Stepwise adjustment of solution conditions: In the pre-mix
container(s)/receiving (post-mix) container embodiment, it should be
noted that the solutions need not be mixed in their entireties in one
step; that is, a portion of the solutions in the pre-mix containers can
be drawn into the receiving container, followed by continued incubation
under pressure of the remaining solutions in the pre-mix containers as
well as in the receiving container. In this manner, stepwise adjustment
of solution conditions can be implemented. In additional embodiments, the
pre-mix containers can have separately actuated valves for addition of
different pre-mix solutions at different points in time. Thus, for
example, for pre-mix containers designated A, B, C, and D, a liquid
sample, such as a protein solution, in pre-mix container A can be
incubated for a period of time, then (with valves to A and B open, but
valves to C and D closed) the contents of pre-mix containers A and B can
be drawn into the receiving container, to alter the original solution
conditions of the liquid sample from container A. After a further period
of incubation, the valve to pre-mix container C can be opened, and the
contents of container C drawn into the receiving container. After yet
another period of incubation, the valve to pre-mix container D can be
opened, and the contents of container D drawn into the receiving
container, followed by still another period of incubation, if desired.
This can be implemented with as many pre-mix containers as desired in
order to adjust the solution conditions of the liquid sample in a
stepwise fashion.

[0094]In the flow-loop embodiment, stepwise adjustment of solution
conditions can be implemented by having several containers, designated,
for example, containers A, B, C, and D. The solutions can be incubated
under high pressure for a period of time. Then valves to containers A and
B can be opened, allowing flow between those containers (and alteration
of the solution conditions of container A as its contents mix with the
contents of container B), while valves to containers C and D can be kept
in a position where flow by-passes containers C and D during an
incubation period. The valves can then be set to allow the contents of
container C to be placed into the flow loop (e.g., by shutting off the
by-pass shunt around container C, and opening the valves to place
container C in the flow loop), where the contents of container C are now
mixed with the contents of containers A and B in the flow loop (and
alteration of the solution conditions of the solution in the flow loop as
its contents mix with the contents of container C), while flow continues
to by-pass container D, for another incubation period. Finally, valves
can be opened to place container D in the flow loop, while shutting off
the by-pass shunt around container D, for yet another adjustment of the
solution conditions of the solution in the flow loop as its contents mix
with the contents of container D, and yet another incubation period. This
can be implemented with as many containers in the flow loop as desired,
with appropriate valves and by-pass shunts, in order to adjust the
solution conditions of the liquid sample in a stepwise fashion.

[0095]These embodiments can be used for refolding of proteins under
various conditions. Lin, U.S. Pat. No. 6,583,268, and Li, M. and Z. Su
(2002), Chromatographia 56(1-2): 33-38, have suggested refolding proteins
at high pH with chaotropes, followed by step-wise reduction of pH,
dilution of the protein solution, and ultrafiltration and gel
chromatography. Using the high-pressure devices as described above,
pressure-modulated refolding (pressures of 250-5000 bar) can be conducted
in non-denaturing chaotrope solutions at alkaline pH (near 10.0) and then
the pH of the solution can be gradually decreased in step-wise fashion
until a value of pH 8.0 is obtained. A rate of 0.2 units per 24 hours,
which would be a period of 10 days to lower the pH from 10 to 8, is
suggested in U.S. Pat. No. 6,583,268; this rate can be adopted as a
general condition, or optimal conditions can be determined on a
protein-by-protein basis. The use of high hydrostatic pressure can reduce
or remove the need to use high concentrations of chaotropes to promote
aggregate dissociation. By combining pressure and chaotrope/pH modulated
refolding methods, higher refolding yields are expected to be achieved.

[0096]In one embodiment, the invention embraces methods of altering
solution conditions under high pressure, comprising the steps of:
providing at least one composition in a solution in at least one first
container; providing at least one agent for changing solution conditions
in at least one additional container, where the contents of the at least
one additional container are not in contact with the contents of the at
least one first container; placing the containers under high pressure;
and causing the contents of the at least one additional container to
contact the contents of the at least one first container, wherein the
contents of the at least one additional container are caused to contact
the contents of the at least one first container over time. In one
embodiment, the contents of the at least one additional container are
caused to contact the contents of the at least one first container in a
continuous manner, whereby the solution conditions of the contents of the
first container are changed continuously over a period of time. In
another embodiment, the contents of the at least one additional container
are caused to contact the contents of the at least one first container in
a continuous manner, whereby the solution conditions of the contents of
the at least one first container are changed step-wise over a period of
time. In one embodiment of this step-wise change in solution conditions,
the pH is changed, and the pH of the contents of the first container is
at about 9 to about 11, or at about 9.5 to about 10.5, or at about 10. In
another embodiment of this step-wise change in solution conditions, the
pH of the contents of the first container is at about 9 to about 11, or
at about 9.5 to about 10.5, or at about 10, and is lowered to a pH of
about 7 to about 8.9, or about 7.5 to about 8.5, or about 8. In another
embodiment of the stepwise method, the pH is lowered by about 0.01 to
about 2 pH units every approximately 24 hours, or by about 0.1 to about 1
pH unit every approximately 24 hours, or by about 0.1 to about 0.5 pH
units every approximately 24 hours, or by about 0.1 to about 0.4 pH units
every approximately 24 hours, or by about 0.1 to about 0.3 pH units every
approximately 24 hours, or by about 0.2 pH units every approximately 24
hours. Incubation periods before, during, and after the solution
condition adjustments can be varied as desired for optimal refolding
yields; for example, incubation under high pressure can be carried out
for a period of any time from about 1 minute to about 1 week, or about 10
minutes to about 48 hours, or about 1 hour to about 48 hours, or about 10
minutes to about 24 hours, or about 1 hours to about 24 hours, or about
10 minutes to about 12 hours, or about 1 hour to about 12 hours, or about
1 hour to about 6 hours prior to adjustment of solution conditions. For
gradual continuous change of solution conditions, the adjustment can be
carried out for a period of any time from about 1 minute to about 1 week,
or about 10 minutes to about 48 hours, or about 1 hour to about 48 hours,
or about 10 minutes to about 24 hours, or about 1 hours to about 24
hours, or about 10 minutes to about 12 hours, or about 1 hour to about 12
hours, or about 1 hour to about 6 hours. For step-wise adjustments of
solution conditions, the interval between adjustments can be for a period
of any time from about 1 minute to about 1 week, or about 10 minutes to
about 48 hours, or about 1 hour to about 48 hours, or about 10 minutes to
about 24 hours, or about 1 hours to about 24 hours, or about 10 minutes
to about 12 hours, or about 1 hour to about 12 hours, or about 1 hour to
about 6 hours. Finally, incubation under high pressure after solution
conditions have been adjusted to the desired end conditions can be
carried out for a period of any time from about 1 minute to about 1 week,
or about 10 minutes to about 48 hours, or about 1 hour to about 48 hours,
or about 10 minutes to about 24 hours, or about 1 hours to about 24
hours, or about 10 minutes to about 12 hours, or about 1 hour to about 12
hours, or about 1 hour to about 6 hours.

[0097]In the method as described above, the contents of the at least one
first container may remain in the first container as the solution
conditions are changed, as would be the case with the
container-in-container embodiment for solution exchange. Alternatively,
all or part of the contents of the at least one first container may no
longer be in the first container as the solution conditions are changed,
as would be the case with the flow-loop or pre-mix container(s)/receiving
(post-mix) container embodiments, in which case the alteration of the
contents of the first container is occurring in a location partly or
entirely apart from the first container. In such a case, it is understood
that reference to changing the solution conditions of the contents of the
at least one first container refers to changing the solution conditions
of the contents that were originally in the at least one first container
(i.e., "the contents of the at least one first container" is understood
to read as "the original contents of the at least one first container
prior to solution exchange").

Introduction of Samples into the Sample Compartments

[0098]Once a suitable material has been chosen for the body of the device,
the samples must be introduced into the sample compartments. The device
is adapted to receive liquid samples, and thus a variety of standard
methods for liquid transfer can be employed. Hand-held or robotic
pipettes, syringes, pumps, and other liquid transfer instruments
well-known in the art can be employed. Care should be taken to exclude as
much residual air as possible from any of the devices prior to
pressurization, which helps prevents material failure and prevents the
oxygen contained in the air from being dissolved in the system. The
devices can be filled in an inert atmosphere, such as nitrogen or argon,
in order to prevent residual air that cannot be excluded from altering
the oxygen content of the liquid when pressure is applied.

[0099]In certain additional embodiments, prior to loading a liquid sample
into the compartment, one or more gases will be sparged through the
sample. Such gases include, but are not limited to, relatively unreactive
gases such as helium, nitrogen, neon, argon, or krypton, where it is
desirable to displace as much dissolved oxygen as possible. Usually
rigorous exclusion of oxygen is desired, but in certain circumstances
where a higher-than-normal oxygen content is desired in the solution, air
or oxygen itself can be sparged through the sample. In yet additional
embodiments, vacuum may be applied to the sample in order to de-gas the
sample. In yet additional embodiments, sparging with unreactive gas can
be followed by vacuum treatment in order to remove as much dissolved
oxygen as possible; the sparge-pump cycle can be repeated as necessary.

Sealing of the Sample Compartments and Sample Introduction

[0100]Several of the devices of the invention provide their own seal,
e.g., the variable loading volume device, which uses a one-way valve plug
for sealing purposes. For devices which do not have their own seal, such
as the 96-well plate, sample compartments can be sealed with seals
fabricated from silicone, rubber or other material. In one embodiment,
the seal material is inert to the contents of the sample well, since the
liquid sample may come into contact with the seal during the experiment.
When a seal such as rubber is used which is not substantially impermeable
to oxygen at high pressure, a second seal which is substantially
oxygen-impermeable at high pressure can applied over the first seal to
reduce or prevent oxygen mass transfer. The one-way valve plug can be
used in a variety of other devices in addition to the variable loading
volume device, such as the 96-well plate (where up to 96 one-way valve
plugs would be used to seal the compartments).

[0101]The sample compartments can be sealed before or after introduction
of the liquid sample. If the sample compartment is sealed after the
introduction of the liquid sample, then the necessity of penetrating the
seal is avoided. However, if the sample compartment is sealed before
introduction of the liquid sample, the seal must allow introduction of
the sample. A seal made of materials such as rubber or silicone can be
pierced with a needle in order to introduce liquid sample; a second
needle can be used to vent air from the compartment. The second, venting
needle is inserted only to the extent needed to penetrate the seal and
minimally extend into the chamber, in order to withdraw as much air as
possible. Filling of the chamber is complete once air is completely
expelled and liquid begins to be expelled from the chamber.

[0102]Since rubber and certain silicones are relatively permeable to
oxygen at high pressure, a second sealing layer can be applied in order
to prevent mass transfer of oxygen at high pressure. A layer of
Mylar® or other suitable material which is substantially
oxygen-impermeable at high pressure can be laid down over the first
seals.

Other Applications

[0103]It should be noted that, while the high-pressure devices have been
discussed above in the context of pharmaceuticals, and in particular for
the refolding of proteins, the application of these devices is not
limited to the pharmaceuticals or protein refolding. The devices can be
used in any applications requiring pressure treatment of samples,
particularly liquid samples. For example, Kunugi et al., Langmuir,
15:4056 (1999) studied temperature and pressure responsive behavior of
thermoresponsive polymers in aqueous solutions at various pressures.
Pressure is well-known to affect chemical reactions; pressure can affect
both reaction kinetics (reactions with negative activation volumes are
accelerated by higher pressure; see Vaneldik et al., Chemical Reviews
89:549 (1989) and Drljaca et al., Chemical Reviews 98:2167 (1998)) and
reaction thermodynamics (transitions which lower system volume are
favored by higher pressure; see J. M. Smith et al., Introduction to
Chemical Engineering Thermodynamics, New York: McGraw-Hill, 2001).

[0104]The invention will be further understood by the following
illustrative examples, which are not intended to limit the invention.

[0105]Solution exchange was studied during pressure treatment with the
solution mixing device described in FIGS. 13-18. A dilution of a known
concentration of Coomassie Blue dye was placed in one pre-mix container
(1.0 ml of 0.015 mg/ml dye). In the other pre-mix sample container, 1 ml
of pure water was placed. Pressure was slowly increased to 2000 bar.
After 10 minutes at this pressure, the high pressure valve connecting to
the side inlet of the chamber is closed and the high pressure syringe is
withdrawn to modulate the piston flow (a calibration was previously
conducted to equate the piston location of the syringe pump relative to
piston location of the pre-mix and receiving solution containers). The
sample was collected and UV/VIS absorbance measured at 570 nm to
determine the final concentration of dye after exchange (FIG. 22). This
data was compared to a standard of Coomassie Blue. Three sequential
experiments were conducted to determine the extent of mixing that
occurred after operating the solution exchange device described in FIGS.
13-18. An absorbance value of 0.55+/-0.5 was measured, corresponding to a
dye concentration of 0.0092 mg/ml dye. A 1:1 dilution of the dye solution
with the pure water, post-mixing, should result in a dye concentration of
0.0075 mg/ml, with an absorbance of 0.43 at 570 nm (FIG. 22). The study
demonstrates that mixing occurred after operating the device three times,
with 1.24 volumes of the solution containing Coommassie Blue dye mixing
with 0.75 volumes of deionized water. This data demonstrates that
solution exchange occurred during pressure treatment.

[0106]This example demonstrates that solution exchange during pressure
treatment alters the refolding and recovery of native protein from
protein aggregates. In previous work, St. John et al. demonstrated that
pressure-induced refolding of protein aggregates can be optimized when
non-denaturing levels of GdnHCl are present during pressure treatment.
St. John et al. showed that lysozyme refolding recoveries increased
linearly from ca. 35% at 0.2M GdnHCl to ca. 80% at 2M GdnHCl after
incubation at 2000 bar for five days (St John, R. J., J. F. Carpenter, et
al. (2002), Biotechnology Progress 18(3): 565-571).

[0107]FIG. 23 shows the results from the current lysozyme refolding
studies where the GdnHCl concentrations were manipulated both before
pressurization to 2000 bar (`no exchange` samples) and during
pressurization (`HP-Exch`). (Atmospheric controls were also run, and
demonstrated that pressure treatment was needed to refold the lysozyme
aggregates.) Lysozyme was refolded with 1M GdnHCl at high pressure (no
solution exchange), resulting in a refolding yield of ca. 53%. Lysozyme
was also refolded at 0.5M GdnHCl at high pressure, without solution
exchange, resulting in a refolding yield of ca. 27%. When lysozyme was
refolded at an initial 1M GdnHCl concentration, followed by solution
exchange and reduction to a 0.5 M GdnHCl concentration during pressure
treatment, a refolding yield of ca. 47% resulted. Thus, while the latter
two experiments both had a final concentration of 0.5M GdnHCl, the
non-exchanged solution had a much lower refolding yield that the
exchanged solution. The non-exchanged lysozyme solution refolded at 1.0M
GdnHCl had a higher refolding yield than either of the solutions ending
at 0.5M GdnHCl.

[0108]High pressure destabilizes hydrophobic and electrostatic contacts
but has very little effect on hydrogen bonding. GdnHCl, on the other
hand, destabilizes hydrogen bonding. Therefore, the addition of
non-denaturing levels of GdnHCl helps facilitate refolding of lysozyme.
During the high pressure solution exchange, the initial higher GdnHCl
concentration (1M) introduces the lysozyme aggregate to a more favorable
environment for aggregate dissociation. Solution exchange under pressure
was then completed to bring the final GdnHCl concentration to 0.5M. As
previously stated, it can be seen that even though the final solution
conditions of both the 0.5 M GdnHCl `no exchange` sample and the solution
exchanged sample are the same, refolding was facilitated in the solution
exchanged sample by the ability to initially start at the higher 1M
chaotrope concentration. The 1M GdnHCl "no exchange" refolding yield
emphasizes that lysozyme remains in the native conformation in the
presence of 1M guanidine, 2000 bar (Randolph, T. W., M. Seefeldt, et al.
(2002), Biochimica Et Biophysica Acta-Protein Structure and Molecular
Enzymology 1595(1-2): 224-234). Consequently, refolding yields of
lysozyme are not decreased by the presence of the high concentration of
chaotrope. Solution exchange during pressure treatment to lower
chaotrope-concentrations can be more beneficial towards increasing yields
for proteins that are more sensitive to the presence of guanidine HCl.
These results show the ability to successfully increase the refolding
yield of a protein aggregate using the technique of solution exchange
during high pressure treatment.

[0109]The experimental conditions used were as follows: An aqueous
suspension of aggregated hen egg white lysozyme was placed in one pre-mix
container with 50 mM Tris-HCl, 1M GdnHCl, 5 mM GSSG, 2 mM DTT at pH 8.0.
A second pre-mix container was filled with 50 mM tris-HCl, 0M GdnHCl, 5
mM GSSG, 2 mM DTT at pH 8.0 containing no protein. The samples were
pressurized over a period of 10 minutes to a final pressure of 2000 bar.
The protein was kept in the dissolution enhancing buffer for 6 hours, at
which point solution exchange was initiated, using the solution exchange
device depicted in FIG. 14. The final combined solution (now in the
receiving container) remained at 2000 bar for another 6 hours before
depressurization. Controls were tested which refolded identical lysozyme
aggregates in solutions containing 50 mM Tris-HCl, 0.5 or 1M GdnHCl, 5 mM
GSSG, 2 mM DTT at pH 8.0, at pressures of 2000 and 1 bar. The sample was
collected from the receiving container and lysozyme catalytic activity
was measured by a method similar to the one described by Jolles (Jolles,
P. (1962). "Lysozymes from Rabbit Spleen and Dog Spleen." Methods of
Enzymology 5: 137).

[0110]The disclosures of all publications, patents, patent applications
and published patent applications referred to herein by an identifying
citation are hereby incorporated herein by reference in their entirety.

[0111]Although the foregoing invention has been described in some detail
by way of illustration and example for purposes of clarity of
understanding, it is apparent to those skilled in the art that certain
minor changes and modifications will be practiced. Therefore, the
description and examples should not be construed as limiting the scope of
the invention.